IMPLEMENTATION OF DIMER-BASED SCREENING SYSTEM IN ESCHERICHIA COLI BL21(DE3) FOR SELECTION OF ACTINOMYCETES COMPOUNDS AS ANTI-HIV CANDIDATE

• Main Author: Permata Sukma, Kenia
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/49510
• Institution: Institut Teknologi Bandung

Anti-retroviral therapy (ART) still experiences limitations in reaching Human Immunodeficiency Virus (HIV) sufferers in Indonesia due to its high price and the emergence of resistance of some strains of the virus. One of the targets of ART therapy is HIV-1 protease, an enzyme with a major role in the process of maturation of the virus thus it can infect new cells. HIV-1 protease contributes to the production all enzymes and structural proteins used to release mature and virulent virions. Actinomycetes are reported to have inhibitory activity against several types of HIV proteases. Therefore, the exploration of Indonesia’s actinomycetes species is expected to be a breakthrough for HIV treatment. In this study, selection of anti-HIV candidate compounds was conducted using a dimer-based screening system on recombinant Escherichia coli BL21(DE3). The construct includes the fusion of the AraC DNA binding domain + HIV-1 protease as the regulator and the green fluorescence protein as the reporter. Confirmation of the plasmid construct was carried out by Polymerase Chain Reaction analysis which showed band electropherograms in the size of ~1076 bp. Sequencing analysis proves 100% similarity and identity between construct used in this study and one previously designed. SDS-PAGE showed the presence of a band in the size of ~24 kDa equal to the size of the fusion protein. Screening of the ability of actinomycetes compounds tested to inhibit HIV protease was carried out on 19 types of actinomycetes compounds. Five of the compounds; BLH 1-12 (2) EA, MAE 1-13 EA, BLH 1-1 EA, BLH 7-5 MetA, LC 98 (1) EA, showed a consistent and significant protease-HIV inhibitory activity at certain concentrations, which was indicated by a greater green fluorescence protein expression compared to the baseline. Thus, in this study, dimer-based screening system method could identify the potential actinomycetes as a new anti-HIV candidate for the protease inhibitor group.